A Dynamic Membrane‐Active Glycopeptide for Enhanced Protection of Human Red Blood Cells against Freeze‐Stress

Abstract

Intracellular delivery of freezing-tolerant trehalose is crucial for cryopreservation of red blood cells (RBCs) and previous strategies based on membrane-disruptive activity usually generate severe hemolysis. Herein, a dynamic membrane-active glycopeptide is developed by grafting with 25% maltotriose and 50% p-benzyl alcohol for the first time to effectively facilitate entry of membrane-impermeable trehalose in human RBCs with low hemolysis. Results of the mechanism acting on cell membranes suggest that reversible adsorption of such benzyl alcohol-grafted glycopeptide on cell surfaces upon weak perturbation with phospholipids and dynamic transition toward membrane stabilization are essential for keeping cellular biofunctions. Furthermore, the functionalized glycopeptide is indicative of typical 𝜶-helical/𝜷-sheet structure-driven regulations of ice crystals during freeze−thaw, thereby strongly promoting efficient cryopreservation. Such all-in-one glycopeptide enables achieving both high cell recovery post-thaw >85% and exceptional cryosurvival >95% in direct freezing protocols. The rationally designed benzyl alcohol-modified glycopeptide permits the development of a competent platform with high generality for protection of blood cells against freeze-stress.

Integration of Trehalose Lipids with Dissociative Trehalose Enables Cryopreservation of Human RBCs

“…The binding of Tre-C n with cell membranes was further examined using ITC, while DOPC was utilized as a lipid model because it is one of the phospholipid components of the outer leaflet in hRBC membranes. , Figure shows the ITC profiles of the interaction of DOPC with trehalose or Tre-C n at 25 °C. Each peak in the upper panel of Figure represents the heat generated by a single titration of the same volume of DOPC into the sample solution, and the lower panel is the plot of fitting curves.…”

“…ITC tests were performed on an ITC200 machine (GE Healthcare) at 25 °C using DOPC as the phospholipid membrane according to refs , . DOPC (2 mM), trehalose lipid, or trehalose (200 μM) solutions were prepared in the mixture of ethanol and DI water (4/1, v/v).…”

Cryoprotectants for red blood cells: Evaluate safety and effectiveness by in vitro measures

“…135,136 The aggregation ability of RBCs was measured by the aggregation index (AI) depending on the kinetics and extent of aggregation, where a larger AI indicates an enhanced ability to aggregate. 109 Gao et al 98 synthesized a dynamic membrane-active glycopeptide called PL-g-(MT/BA) (PMB) to improve RBC cryopreservation, which could efficiently help nonpermeable trehalose enter human RBCs with low hemolysis. In addition to high trehalose uptake, maintaining the biofunctions of human RBCs is crucial during incubation and freeze-thaw.…”

A biocompatible cell cryoprotectant based on sulfoxide-containing amino acids: mechanism and application

“…Recently, a variety of CPAs have been developed to eliminate the dependence on glycerol or DMSO, including sugars (e.g. trehalose, sucrose), [24][25][26][27] alcohols (e.g. ethyleneglycol, propylene glycol), 28 antifreeze proteins and their mimics, 29,30 and osmoprotectants (e.g.…”